S. pneumoniae (S.p.) is an important human pathogen causing more than 1 million deaths per year world-wide. In addition, S.p is responsible for an estimated 7 million cases of otitis media each year in the US alone. The currently available conjugate vaccine includes 7 of the 90 known capsule types. While this vaccine has significantly reduced invasive disease and colonization caused by vaccine serotypes, there has been an unexpected increase in both invasive disease and colonization caused by non-vaccine serotypes. In addition, the vaccine does not effectively protect against otitis media. Colonization of the respiratory tract is an essential precursor to disease. Despite the importance of adherence to colonization and, therefore, pathogenesis, the mechanisms by which S.p adheres to airway epithelia remain unclear. We have identified a novel exoglycosidase-dependent adherence mechanism requiring the S.p. neuraminidase, NanA, and 2- galactosidase, BgaA. This mechanism is relevant to adherence of recent clinical isolates and S.p. adherence to human primary epithelial cells, suggesting that it will be relevant in vivo. Our preliminary data demonstrate that while NanA exposes a receptor for adherence, BgaA acts as an adhesin. To further define this mechanism of adherence we have 2 Specific Aims: 1) Elucidate the BgaA binding site. We will define the BgaA region that mediates adherence to human epithelial cells. BgaA is the 2nd largest protein expressed by S.p. and possesses a C-terminal region of unknown function. We will perform molecular and structural analyses to elucidate how BgaA acts as an adhesin. Different regions of the protein will be expressed and used in binding and inhibition studies to identify the region(s) that mediate(s) adherence. The residues that contribute to binding will subsequently be identified by mutagenesis scanning. Structuring of BgaA will be utilized as an alternative approach to identify the binding site and identify the function of the C-terminal region of the protein. 2) Identify the BgaA receptor on the epithelial cell surface. The initial attachment of S.p. has been proposed to occur through a glycan receptor exposed by neuraminidase; however, the adhesin is unknown and there have been conflicting reports of different receptors. We will use a comprehensive range of techniques to determine if BgaA binds to a glycan and if this is structure in the context of a protein or lipid. Following identification of the receptor we will further characterize its interaction with BgaA. The successful completion of these Specific Aims will provide an increased understanding of this mechanism of S.p. adherence. This knowledge is likely to contribute significantly to the long-term goal of developing more effective vaccines and/or treatments to reduce the burden of S.p. disease. PUBLIC HEALTH RELEVANCE: Colonization of the human airway is an essential precursor to pneumococcal disease; however the mechanisms by which the bacteria initially attach to the airway are poorly understood. This study will characterize a novel glycosidase-dependent mechanism of adherence. We will identify the bacterial adhesin and the receptor on the epithelial surface. Our increased understanding of pneumococcal adherence will help us achieve the longer term goal of developing a more effective vaccine or treatment to reduce the burden of pneumococcal disease.